The ferritins constitute a class of iron storage proteins widely distributed among animals, plants, and bacteria. Iron is stored within these proteins in the form of a hydrous ferric oxide mineral core. The mechanisms by which iron is acquired and released by ferritins are unknown. The proposed research is intended to answer this important question. The binding and translocation of Fe(II) and Fe(III) during the early stage of iron deposition will be studied by Mossbauer, electron paramagnetic resonance (EPR), electron nuclear double resonance (ENDOR), and electron spin echo envelope modulation (ESEEM) spectroscopies. Characterization of mixed-valence Fe(II)-Fe(III) cluster complexes, the location of the iron binding sites, and the elucidation of the ligand environment of the metal will receive special attention. The midpoint reduction potentials of iron- apoferritin species generated in the reaction prior to core development will be determined in order to characterize the redox chemistry of the protein. Studies of flavin penetration of the protein shell and the possible role of the hydrophobic channels as pathways for oxygen diffusion will also be carried out. Cr(III) will be used as a kinetically inert probe of iron binding and deposition in apoferritin. Various mixed metal apoferritin complexes expected to have novel structural and magnetic properties will be prepared with Cr(III), Cu(II), Mn(II), VO(IV), and Fe(III). Extended x-ray absorption fine structure (EXAFS) and x- ray crystallographic measurements on various complexes will be carried out to locate the metal binding sites and to determine metal-metal distances. Radicals generated during iron deposition in ferritin will be identified and their possible role in protein damage assessed. From this research a picture should emerge of the sequence of events which lead to formation of the polynuclear iron core in ferritin, providing a greater appreciation of the special function of this protein in the metabolism of iron.